Recognition Events Research Articles

Live-Cell-Templated Dynamic Combinatorial Chemistry.

Dynamic covalent chemistry combines in a single step the screening and synthesis of ligands for biomolecular recognition. In order to do that, a chemical entity is used as template within a dynamic combinatorial library of interconverting species, so that the stronger binders are amplified due to the efficient interaction with the target. Here we employed whole A549 living cells as template in a dynamic mixture of imines, for which amplification reflects the efficient and selective interaction with the corresponding extracellular matrix. The amplified polyamine showed strong interaction with the A549 extracellular matrix in on-cell NMR experiments, while combination of NMR, SPR, and molecular dynamics simulations in model systems provided insights on the molecular recognition event. Notably, our work pioneers the use of whole living cells in dynamic combinatorial chemistry, which paves the way towards the discovery of new bioactive molecules in a more biorelevant environment.

Live‐Cell‐Templated Dynamic Combinatorial Chemistry

AbstractDynamic covalent chemistry combines in a single step the screening and synthesis of ligands for biomolecular recognition. In order to do that, a chemical entity is used as template within a dynamic combinatorial library of interconverting species, so that the stronger binders are amplified due to the efficient interaction with the target. Here we employed whole A549 living cells as template in a dynamic mixture of imines, for which amplification reflects the efficient and selective interaction with the corresponding extracellular matrix. The amplified polyamine showed strong interaction with the A549 extracellular matrix in on‐cell NMR experiments, while combination of NMR, SPR, and molecular dynamics simulations in model systems provided insights on the molecular recognition event. Notably, our work pioneers the use of whole living cells in dynamic combinatorial chemistry, which paves the way towards the discovery of new bioactive molecules in a more biorelevant environment.

Signal amplification based on tannic acid-assisted cyclic conversion of Fe(III)/Fe(II) for ultrasensitive electrochemical immunoassay of CA 12-5

A signal amplification protocol based on tannic acid (TA)-assisted cyclic conversion of Fe(III)/Fe(II) was designed for highly sensitive immunoassay of carbohydrate antigen 12-5 (CA12-5). Initially, graphene oxide-methylene blue (GO-MB) and ionic liquid functionalized reduced graphene oxide-Au (IL-rGO-Au) composite were sequentially covered on the surface of electrode as stable signal substrate. The labeling antibody/Au-reduced graphene oxide/zeolitic imidazolate framework-metal polyphenol network (Ab2/Au-rGO/ZIF-MPN) as novel probe was prepared by firstly coating TA on ZIF-8 to form ZIF-TA, then adsorbing a certain amount of Fe(III) to form intense metal polyphenol network (MPN) as the coating of probe on account of combination of TA and Fe(III), finally adsorbing Au-rGO composite on ZIF-MPN for immobilization of Ab2. In the existence of target CA12-5, a sandwiched electrochemical immunosensor was successfully constructed via specific recognition events between antigen and antibody. With acidic H2O2 stimulation, the coating of probe was cracked into TA and Fe(III), then TA reduced Fe(III) into Fe(II) with significant catalytic capacity, which reacted with H2O2 to activate Fenton reaction and meanwhile produced Fe(III) and hydroxyl radicals (·OH). Subsequently, the gained Fe(III) was reduced to Fe(II) again by TA, forming the cyclic conversion of Fe(III)/Fe(II) to produce adequate ·OH for degradation of MB. The tactic averted the employment of vulnerable enzymes and noble metal materials, meanwhile achieving conspicuous assay performance with a linear range from 0.00005–500 U mL−1 and ultralow detection limit of 2.3 × 10−3 mU mL−1 under optimal conditions.

Encoding quantized fluorescence states with fractal DNA frameworks

Signal amplification in biological systems is achieved by cooperatively recruiting multiple copies of regulatory biomolecules. Nevertheless, the multiplexing capability of artificial fluorescent amplifiers is limited due to the size limit and lack of modularity. Here, we develop Cayley tree-like fractal DNA frameworks to topologically encode the fluorescence states for multiplexed detection of low-abundance targets. Taking advantage of the self-similar topology of Cayley tree, we use only 16 DNA strands to construct n-node (n = 53) structures of up to 5 megadalton. The high level of degeneracy allows encoding 36 colours with 7 nodes by site-specifically anchoring of distinct fluorophores onto a structure. The fractal topology minimises fluorescence crosstalk and allows quantitative decoding of quantized fluorescence states. We demonstrate a spectrum of rigid-yet-flexible super-multiplex structures for encoded fluorescence detection of single-molecule recognition events and multiplexed discrimination of living cells. Thus, the topological engineering approach enriches the toolbox for high-throughput cell imaging.

Deleterious effects of carbon-carbon dipolar coupling on RNA NMR dynamics.

Many regulatory RNAs undergo dynamic exchanges that are crucial for their biological functions and NMR spectroscopy is a versatile tool for monitoring dynamic motions of biomolecules. Meaningful information on biomolecular dynamics requires an accurate measurement of relaxation parameters such as longitudinal (R1) rates, transverse (R2) rates and heteronuclear Overhauser effect (hNOE). However, earlier studies have shown that the large 13C-13C interactions complicate analysis of the carbon relaxation parameters. To investigate the effect of 13C-13C interactions on RNA dynamic studies, we performed relaxation measurements on various RNA samples with different labeling patterns and compared these measurements with the computational simulations. For uniformly labeled samples, contributions of the neighboring carbon to R1 measurements were observed. These contributions increased with increasing magnetic field and overall correlation time ([Formula: see text]) for R1 rates, necessitating more careful analysis for uniformly labeled large RNAs. In addition, the hNOE measurements were also affected by the adjacent carbon nuclei. Unlike R1 rates, R1ρ rates showed relatively good agreement between uniformly- and site-selectively labeled samples, suggesting no dramatic effect from their attached carbon, in agreement with previous observations. Overall, having more accurate rate measurements avoids complex analysis and will be a key for interpreting 13C relaxation rates for molecular motion that can provide valuable insights into cellular molecular recognition events.

(Invited) Plasmon Enhanced IR Spectroscopy and Electrochemistry

The localized surface plasmon resonance (LSPR) arises from the collective oscillation of conduction electrons of metal nanostructures which can be used to monitor recognition events of biomolecules at single nanoparticles1. The enhanced electric field around the nanostructures due to LSPR will significantly enhance the Raman scattering, fluorescence and IR spectra, which enable the realization of single molecule detection. In addition, the LSPR will excite high-energy electron-hole pair (referred to as “hot electrons” and “hot holes”) emerging on metal surface. The energetic charges will considerably affect the electrochemical reactions occurring at the nanoparticles. When the plasmonic metallic nanostructures are coupled to other substrates, for example, the semiconductor (i.e., TiO2, MoS2), the plasmon-excited hot electron-hole at nanoparticle surface can communicate with the conductance and valence bands of the semiconductors, resulting in variation in electro/photocatalytic activity. In this talk, we will start with the LSPR enhanced IR for biosensing.1 Then, we will show how the LSPR accelerates electrochemical reactions of electroactive biomolecules such as glucose on gold nanoparticles and hydrogen evolution reaction at molybdenum disulphide nanosheets.2 Based on the plasmonics accelerated electrochemical reactions (PAER), high sensitive electrochemical biosensors for detection of glucose and other electroactive biomolecules have been constructed.

Configurational Selection in Azobenzene-Based Supramolecular Systems Through Dual-Stimuli Processes.

Azobenzene is one of the most studied light‐controlled molecular switches and it has been incorporated in a large variety of supramolecular systems to control their structural and functional properties. Given the peculiar isomeric distribution at the photoexcited state (PSS), azobenzene derivatives have been used as photoactive framework to build metastable supramolecular systems that are out of the thermodynamic equilibrium. This could be achieved exploiting the peculiar E/Z photoisomerization process that can lead to isomeric ratios that are unreachable in thermal equilibrium conditions. The challenge in the field is to find molecular architectures that, under given external circumstances, lead to a given isomeric ratio in a reversible and predictable manner, ensuring an ultimate control of the configurational distribution and system composition. By reviewing early and recent works in the field, this review aims at describing photoswitchable systems that, containing an azobenzene dye, display a controlled configurational equilibrium by means of a molecular recognition event. Specifically, examples include programmed photoactive molecular architectures binding cations, anions and H‐bonded neutral guests. In these systems the non‐covalent molecular recognition adds onto the thermal and light stimuli, equipping the supramolecular architecture with an additional external trigger to select the desired configuration composition.

Magic-Angle-Pulse Driven Separation of Degenerate 1 H Transitions in Methyl Groups of Proteins: Application to Studies of Methyl Axis Dynamics.

Dynamics of protein side chains is one of the principal determinants of conformational entropy in protein structures and molecular recognition events. We describe NMR experiments that rely on the use of magic-angle pulses for efficient isolation of degenerate 1 H transitions of the I=3/2 manifold of 13 CH3 methyl groups, and serve as 'building blocks' for the measurement of transverse spin relaxation rates of the fast- and slow-relaxing 1 H transitions - the primary quantitative reporters of methyl axis dynamics in selectively {13 CH3 }-methyl-labelled, highly deuterated proteins. The magic-angle-pulse driven experiments are technically simpler and, in the absence of relaxation, predicted to be 2.3-fold more sensitive than previously developed analogous schemes. Validation of the methodology on a sample of {13 CH3 }-labeled ubiquitin demonstrates quantitative agreement between order parameters of methyl three-fold symmetry axis obtained with magic-angle-pulse driven experiments and other established NMR techniques, paving the way for studies of methyl axis dynamics in human DNAJB6b chaperone, a protein that undergoes exchange with high-molecular-weight oligomeric species.

Voltammetric detection of miRNA hybridization based on electroactive indicator-cobalt phenanthroline

The indicator-based nucleic acid detection protocol is one of the major approaches to monitor the sequence-selective nucleic acid hybridization-mediated recognition events in biochemical analysis. The metal complex, cobalt phenanthroline, [Co(phen)33+], which is one of the electroactive indicators, interacts more with double stranded nucleic acids via intercalation. Thus, this interaction permits an increase at the electrochemical signal of [Co(phen)33+]. In our study, the interaction of metal complex, [Co(phen)33+] with nucleic acids was examined using pencil graphite electrodes (PGEs) in combination with differential pulse voltammetry (DPV) technique. The voltammetric detection of miRNA-34a was investigated based on the changes at the electrochemical signal of [Co(phen)33+] under optimized experimental conditions; such as accumulation potentialof metal complex and DNA probe concentration, hybridization time, target miRNA concentration. Furthermore, the selectivity of electrochemical miRNA-34a biosensor was studied in contrast to different miRNAs. The applicability of indicator-based biosensor specific to miRNA-34a was also presented by using total RNA samples.

Tuning Supramolecular Selectivity for Hydrosulfide: Linear Free Energy Relationships Reveal Preferential C-H Hydrogen Bond Interactions.

Supramolecular anion receptors can be used to study the molecular recognition properties of the reactive yet biologically critical hydrochalcogenide anions (HCh-). Achieving selectivity for HCh- over the halides is challenging but necessary for not only developing future supramolecular probes for HCh- binding and detection, but also for understanding the fundamental properties that govern these binding and recognition events. Here we demonstrate that linear free energy relationships (LFERs)-including Hammett and Swain-Lupton plots-reveal a clear difference in sensitivity to the polarity of an aryl C-H hydrogen bond (HB) donor for HS- over other HCh- and halides. Analysis using electrostatic potential maps highlights that this difference in sensitivity results from a preference of the aryl C-H HB donor for HS- in this host scaffold. From this study, we demonstrate that LFERs are a powerful tool to gain interpretative insight into motif design for future anion-selective supramolecular receptors and highlight the importance of C-H HB donors for HS- recognition. From our results, we suggest that aryl C-H HB donors should be investigated in the next generation of HS- selective receptors based on the enhanced HS- selectivity over other competing anions in this system.

Resolving Binding Events on the Multifunctional Human Serum Albumin.

Physiological processes rely on initial recognition events between cellular components and other molecules or modalities. Biomolecules can have multiple sites or mode of interaction with other molecular entities, so that a resolution of the individual binding events in terms of spatial localization as well as association and dissociation kinetics is required for a meaningful description. Here we describe a trichromatic fluorescent binding‐ and displacement assay for simultaneous monitoring of three individual binding sites in the important transporter and binding protein human serum albumin. Independent investigations of binding events by X‐ray crystallography and time‐resolved dynamics measurements (switchSENSE technology) confirm the validity of the assay, the localization of binding sites and furthermore reveal conformational changes associated with ligand binding. The described assay system allows for the detailed characterization of albumin‐binding drugs and is therefore well‐suited for prediction of drug‐drug and drug‐food interactions. Moreover, conformational changes, usually associated with binding events, can also be analyzed.

Signal-off tuned signal-on (SF-T-SN) colorimetric immunoassay for amantadine using activity-metalmodulated peroxidase-mimicking nanozyme

Immunoassays facilitate a wide range of rapid, simple and sensitive biochemical detection analyses. We developed a signal-off tuned signal-on (SF-T-SN) colorimetric immunoassay for the detection of the antiviral drug, amantadine (AMD). Using this strategy, the conventional competitive signal-off immunoassay acts as a foundation to induce different immune recognition events in the absence and presence of AMD. The introduction of activity-metal modulated peroxidase-mimicking nanozymes converts the conventional signal-off model to a signal-on model, thereby facilitating drug detection, and overcoming disadvantages with natural enzymes. Importantly, due to switching of the reporting mechanism, and the high catalytic activity of the nanozyme, this “off-to-on” pathway makes the nanozyme-based colorimetric signal proportional to AMD concentrations, thereby circumventing the inherent drawbacks of limited signaling abilities and sensitivities with conventional signal-off immunoassay-based small molecule detection systems. AMD concentrations can be read by the eye or by the optical signals. Based on this protocol, system sensitivity (0.195 ng mL−1 AMD for the naked eye, and 0.134 ng mL−1 AMD for optical detection) not only outperformed reported methods, but it dramatically improved the naked eye observations and UV–vis measurements, when compared with conventional signal-off immunoassays. AMD concentrations spiked into chicken samples were also detected with acceptable recoveries, and relative standard derivations (RSD), indicating this method is highly applicable for biochemical analyses and food safety monitoring.

Light-Harvesting Nanoparticle Probes for FRET-Based Detection of Oligonucleotides with Single-Molecule Sensitivity.

Controlling the emission of bright luminescent nanoparticles by a single molecular recognition event remains a challenge in the design of ultrasensitive probes for biomolecules. Herein, we developed 20-nm light-harvesting nanoantenna particles, built of a tailor-made hydrophobic charged polymer poly(ethyl methacrylate-co-methacrylic acid), encapsulating circa 1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87-fold brighter than quantum dots QDots 605 in single-particle microscopy (with 550-nm excitation), these DNA-functionalized nanoparticles exhibit over 50 % total FRET efficiency to a single hybridized FRET acceptor, a highly photostable dye (ATTO665), leading to circa 250-fold signal amplification. The obtained FRET nanoprobes enable single-molecule detection of short DNA and RNA sequences, encoding a cancer marker (survivin), and imaging single hybridization events by an epi-fluorescence microscope with ultralow excitation irradiance close to that of ambient sunlight.

Light‐Harvesting Nanoparticle Probes for FRET‐Based Detection of Oligonucleotides with Single‐Molecule Sensitivity

AbstractControlling the emission of bright luminescent nanoparticles by a single molecular recognition event remains a challenge in the design of ultrasensitive probes for biomolecules. Herein, we developed 20‐nm light‐harvesting nanoantenna particles, built of a tailor‐made hydrophobic charged polymer poly(ethyl methacrylate‐co‐methacrylic acid), encapsulating circa 1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87‐fold brighter than quantum dots QDots 605 in single‐particle microscopy (with 550‐nm excitation), these DNA‐functionalized nanoparticles exhibit over 50 % total FRET efficiency to a single hybridized FRET acceptor, a highly photostable dye (ATTO665), leading to circa 250‐fold signal amplification. The obtained FRET nanoprobes enable single‐molecule detection of short DNA and RNA sequences, encoding a cancer marker (survivin), and imaging single hybridization events by an epi‐fluorescence microscope with ultralow excitation irradiance close to that of ambient sunlight.

Exploring the RNA-Recognition Mechanism Using Supervised Molecular Dynamics (SuMD) Simulations: Toward a Rational Design for Ribonucleic-Targeting Molecules?

Although proteins have represented the molecular target of choice in the development of new drug candidates, the pharmaceutical importance of ribonucleic acids has gradually been growing. The increasing availability of structural information has brought to light the existence of peculiar three-dimensional RNA arrangements, which can, contrary to initial expectations, be recognized and selectively modulated through small chemical entities or peptides. The application of classical computational methodologies, such as molecular docking, for the rational development of RNA-binding candidates is, however, complicated by the peculiarities characterizing these macromolecules, such as the marked conformational flexibility, the singular charges distribution, and the relevant role of solvent molecules. In this work, we have thus validated and extended the applicability domain of SuMD, an all-atoms molecular dynamics protocol that allows to accelerate the sampling of molecular recognition events on a nanosecond timescale, to ribonucleotide targets of pharmaceutical interest. In particular, we have proven the methodological ability by reproducing the binding mode of viral or prokaryotic ribonucleic complexes, as well as that of artificially engineered aptamers, with an impressive degree of accuracy.

Lectins-Robust and Quintessential Proteins of Nature

Recent advances in glycobiology have proclaimed that cell surface saccharides play a pivotal role in recognition events. More accurately, these saccharides may be complexed by lectins. Lectins are saccharide binding proteins that are highly specific for sugar moieties of other molecules. They are ubiquitous in nature. They are diverse proteins with spectacular potential and overwhelming medical applications. Hence, there is quest for identification of new lectins and their precise mechanism of action with specific ligands. In this review, main features of lectins are succinctly described with emphasis on ligand binding and therapeutic applications.

Smartphone-assisted robust enzymes@MOFs-based paper biosensor for point-of-care detection

Portable devices featured with fast analysis and affordable methodologies for clinical diagnostics have stimulated the rapid development of point-of-care (POC) technologies, potentially lowering the mortality rate. Herein, we demonstrated a portable, robust, and user-friendly intelligent metal-organic frameworks (MOFs) paper device, called smartphone-assisted biomimetic MOFs nanoreactor colorimetric paper (SBMCP), for on-demand POC detection of endogenous biomolecules. The concept of this paper platform was analogous to the intracellular cascades signal transduction, wherein the single/multiple enzymes components trapped within a ZIF-8 exoskeleton allowed the sensitive and selective recognition of target analyte via the accessible micropores network of ZIF-8, and then transferred the recognition event to a visual color signal based on the cascade reaction. Meanwhile, the ZIF-8 exoskeleton also endowed the enzymes with significantly elevated stability. As a result, this robust and portable SBMCP sensor enabled the on-site analysis of different important disease-related biomolecules through modulating the enzyme cascades, combining with a custom-designed smartphone application for signal readout. In the SBMCP assay, no sophisticated instruments or professional skill of the user was required, only 5μL sample volume was needed, and the whole analysis process could be achieved within a portable MOFs paper and pervasive smartphone, endowing this new assay with the merits of low-cost, time-saving and easy-to-use. We demonstrated this SBMCP sensor was capable of real-time colorimetric detection of glucose and uric acid in diabetes and gout events. It is believed that this portable biosensor platform proposed herein potentially represents promising alternatives for POC diagnosis, especially applicable in developing world and resource-limitedsettings.

Molecularly Imprinted Polymers and Surface Imprinted Polymers Based Electrochemical Biosensor for Infectious Diseases.

Owing to their merits of simple, fast, sensitive, and low cost, electrochemical biosensors have been widely used for the diagnosis of infectious diseases. As a critical element, the receptor determines the selectivity, stability, and accuracy of the electrochemical biosensors. Molecularly imprinted polymers (MIPs) and surface imprinted polymers (SIPs) have great potential to be robust artificial receptors. Therefore, extensive studies have been reported to develop MIPs/SIPs for the detection of infectious diseases with high selectivity and reliability. In this review, we discuss mechanisms of recognition events between imprinted polymers with different biomarkers, such as signaling molecules, microbial toxins, viruses, and bacterial and fungal cells. Then, various preparation methods of MIPs/SIPs for electrochemical biosensors are summarized. Especially, the methods of electropolymerization and micro-contact imprinting are emphasized. Furthermore, applications of MIPs/SIPs based electrochemical biosensors for infectious disease detection are highlighted. At last, challenges and perspectives are discussed.

Critical parameters in surface plasmon resonance biosensor development: The interaction between estrogen receptor and estrogen response element as model

Estrogenic compounds are contaminants that may be active at low concentrations and are a major concern for environmental quality. They interact with organisms via Estrogen Receptors (ER). Some detection methods which have been developed use the ability of ER to interact with short consensus DNA sequences known as Estrogen Response Elements (ERE). Surface Plasmon Resonance (SPR) based techniques allow detection of interaction without labelled molecule use. Such optical transductors are widely used to convert the biological recognition signals into electric quantifiable signals. In this study, SPR is used to assess signal variation in the presence of estrogenic compounds. The combination of physical properties and biological recognition events (e.g. ER/ERE) permits the development of biosensors. These require several steps: activation of the surface, DNA sequence binding, ERE sequence evaluation, ER preparation, characterization of binding properties and regeneration of the surface. This article focuses on the mode of surface activation, protein-DNA binding conditions and the regeneration of ERE. After giving a summary of the literature concerning the usual conditions employed in these steps, an evaluation of some key parameters is given.

Mannan detecting C-type lectin receptor probes recognise immune epitopes with diverse chemical, spatial and phylogenetic heterogeneity in fungal cell walls.

During the course of fungal infection, pathogen recognition by the innate immune system is critical to initiate efficient protective immune responses. The primary event that triggers immune responses is the binding of Pattern Recognition Receptors (PRRs), which are expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). However, the precise spatial distribution of the ligands that induce immune responses within the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate recognition domains (CRDs) 4–7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and of the β-1,3 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall of fungi grown in vitro in rich and minimal media. We show that epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic relationships between fungi, and that Fc-lectin binding discriminated between mannosides expressed on different cell morphologies of the same fungus. We also demonstrate CTL epitope differentiation during different phases of the growth cycle of Candida albicans and that MR and DC-SIGN labelled outer chain N-mannans whilst dectin-2 labelled core N-mannans displayed deeper in the cell wall. These immune receptor maps of fungal walls of in vitro grown cells therefore reveal remarkable spatial, temporal and chemical diversity, indicating that the triggering of immune recognition events originates from multiple physical origins at the fungal cell surface.